High radiance illumination sources are required for fluorescence imaging and analysis, including fluorescence microscopy. Some applications require broadband or white light illumination. Other applications require relatively narrow band illumination of a particular wavelength range in the ultraviolet (UV), visible or infrared (IR) spectral region.
For example, conventional microscopy illumination systems typically utilize short arc lamps such as high pressure mercury, metal halide, and xenon lamps. These lamps are capable of very high radiance and are suitable sources for direct coupled illumination systems, as well as light guide coupled illumination systems, e.g. using a liquid light guide or a fiber light guide. Nevertheless, it is recognized that there are a number of problems associated with conventional lamp technologies, such as short lifetime, temporal variation of the output power, high voltage operation (typically kilovolts are required to strike the lamp), and use of mercury. The latter is now seen as an environmental hazard and subject to regulations to limit use in numerous countries throughout the world.
Solid state light lighting technology has progressed significantly in recent years and some high brightness light sources using solid state Light Emitting Devices (LEDs), e.g. light emitting diodes, are now available that can potentially provide sufficiently high radiance, broadband illumination for replacement of conventional arc lamps. Solid state LED light sources can offer advantages over conventional arc lamps, such as, much improved lifetime, lower cost of ownership, lower voltage operation, lower power consumption (enabling some battery operated portable devices), and freedom from mercury. Additionally LED light sources can be readily controlled electronically, by modulating the current or voltage driving the device, which allows for fast switching and intensity control through the LED driver, which can be a significant advantage in many applications.
Nevertheless, despite technological advances in LED technology, high brightness LED light sources are not available to cover all wavelengths required for illumination systems for fluorescence imaging and analysis. In particular, the output of LED devices still do not match the radiance of traditional arc-lamps in some regions of the visible spectrum, especially in the 540 nm to 630 nm spectral band, i.e. in the green/yellow/amber range of the visible spectrum. The solid state lighting industry refers to this issue as the “green gap”. Emission in this region of the spectrum is fundamentally limited by the lack of availability of semiconductor materials having a suitable band gap to produce light of the required wavelength.
This is a particular problem for fluorescence imaging and analysis which may, for example, require illumination of a biological sample with a relatively narrow band of illumination of a particular wavelength that is absorbed by a selected fluorophore or marker in the substance under test.
For example, a traditional fluorescence illumination system, e.g. for fluorescence imaging or microscopy, comprises a short arc mercury lamp which provides light emission having spectral peaks near 365 nm, 405 nm, 440 nm, 545 nm and 575 nm. Standard fluorophores that are commonly used for fluorescence imaging and analysis are selected to have absorption spectra having peaks optimized to match these lamp emission peaks. To replace a standard mercury lamp illuminator with a LED based illuminator, it is desirable to be able to provide emission at the same wavelengths and with a comparable output power. There are suitably powerful LEDs that are commercially available for emission at 365 nm, 405 nm, 440 nm. However, in view of the “green gap” mentioned above, there are currently no single color, high brightness LEDs commercially available for emission at 545 nm and 575 nm.
It is well known in the art of LED lighting and illumination to use LEDs in combination with luminescent materials, i.e. fluorescent materials or phosphors, to generate light of wavelengths that are outside the range emitted directly by the LEDS, i.e. by wavelength conversion. In particular, a UV or blue light emitting LED may be combined with a remote or direct die-contact phosphor layer or coating to obtain broadband light emission of a desired color temperature. For example, a blue light emitting diode or diode array with an emission peak in the range between 445 nm and 475 nm is combined with a phosphor layer comprising particles of Ce:YAG (cerium doped yttrium aluminum garnet) suspended in an encapsulant material such as silicone, which is deposited directly on the LED. The blue light from the LED is absorbed by the phosphor and generates a broadband green/yellow/amber light which combines with the scattered blue light to produce a spectrum that provides the perception of white light. The overall brightness is limited by the blue light intensity from the LED and thermal quenching of the phosphor, and the spectrum provides limited emission in regions of the spectrum seen as green/yellow, approximately 560 nm and amber, approximately 590 nm.
Thus, relative to a mercury lamp, commercially available white light LEDs that use a blue light emitting LED combined with a Ce:YAG phosphor, produce significantly weaker emission in the 545 nm and 575 nm regions. For example, at the objective plane of a microscope, output power at 545 nm and 575 nm from such a white light LED was found to be about 10 times lower than the output power from a mercury lamp. This level of power is insufficient for most conventional fluorescence microscopy applications.
By increasing the drive current, some improvement of the light output can be achieved, but fundamentally, the power in this circumstance is limited by the maximum drive current density (i.e. current per unit area) and factors, such as, the LED optical to electrical conversion efficiency, the LED output intensity, the phosphor quantum efficiency, and thermal quenching of both the LED and phosphor, as well as the cooling capacity. Even in the best case, the output from an overdriven air cooled white LED is still 4 to 5 times less than a conventional lamp within the 545 nm and 575 nm spectral range and the lifetime may be significantly reduced by overdriving the device.
The following references provide some other examples of the use of LED sources combined with fluorescent materials or phosphors in other forms.
U.S. Pat. No. 7,898,665 to Brukilacchio et al., issued Mar. 1, 2011, entitled “Light Emitting Diode Illumination System,” for example, discloses a system comprising an arrangement of multiple LEDS that are coupled to a fluorescent rod which emits at a different wavelength to provide sufficiently high brightness illumination for applications such as microscopy or endoscopy. For example a single crystal of Ce:YAG may be pumped by multiple LEDs to generate yellow or amber emission. However, the efficiency of such a device would be limited by total internal reflection due to the high index of refraction of Ce:YAG and requires coupling of multiple LEDs to generate output of sufficient brightness, which increases the cost, size, thermal and electrical requirements.
To provide a more compact and efficient system, the above referenced related copending U.S. Patent application No. 61/651,130, discloses an illumination system that comprises a laser light source, e.g. providing blue light emission in the 440 nm to 490 nm range, for excitation of a wavelength conversion module comprising a wavelength conversion medium, such as Ce:YAG crystal, of a particular shape and size, set in a mounting for thermal dissipation, and an optical concentrator. The shape and size of the wavelength conversion crystal, provides a compact light source with a configuration suitable for applications that require high brightness and narrow bandwidth illumination at a selected wavelength, e.g. for fluorescence microscopy, or other applications requiring étendue-limited coupling or light guide coupling. While effective, due to the particular shape and size of the crystal and cooling requirements, this system is currently relatively costly to manufacture. A solution that is lower cost, compact and provides a broader spectrum is desirable for some applications.
Thus, there is a need for improved or alternative high radiance illumination sources, particularly those that can provide illumination at wavelengths of 545 nm and 575 nm, for example, for fluorescence imaging and analysis applications.